Culture chamber and culture method

ABSTRACT

Provided is a culture chamber that includes a plurality of recesses each formed of a bottom portion and an opening portion. The bottom portion has a hemispherical shape or a truncated cone shape. The opening portion is defined by a wall that surrounds an area from a boundary between the opening portion and the bottom portion to an end of each of the recesses, the wall having a taper angle in a range from 1 to 20 degrees. An equivalent diameter of the boundary is from 50 μm to 2 mm and a depth from a bottom of the bottom portion to the end of each of the recesses is from 0.6 or more times to 3 or less times the equivalent diameter, and the wall defining the opening portion forms a surface continuous to the bottom portion. An inclination of the continuous surface changes at the boundary.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/896,251, filed Dec. 4, 2015, which is based upon and claims thebenefit of priority from Japanese patent application No. 2013-120915,filed on Jun. 7, 2013, the disclosures of which are incorporated byreference herein in their entireties.

FIELD

The present invention relates to culture of cells and harvesting of thecells.

BACKGROUND

Along with the recent development of cell technology, new culturemethods to obtain cells having a function similar to an in-vivo functionby mimicking an in-vivo pericellular environment or morphology have beendeveloped. An attempt has been made to use cells cultured by suchmethods as a simulator for treatment or biological reaction. Variousculture methods have been developed, such as a method of culturing cellsusing a culture support composed of a sponge or fiber; a suspensionculture method in which cells are suspended in a medium so that thecells spontaneously form a spheroid; and a method of culturing cells toform a spheroid by performing a cell non-adhesion treatment on aconventional culture chamber (a flask or the like). In particular, aspheroid culture is an excellent method by which interactions of cellscan be maintained, and thus the method is applied to various cells suchas pancreatic islet cells, liver cells, stem cells, and cancer cells. Inrecent years, studies focusing on the size of a spheroid have been made.For example, in a drug screen test using cancer cells, the diameter orvolume of a spheroid is used as an index (see, Juergen Friedrich1, etal., “Spheroid-based drug screen: considerations and practicalapproach”, PROTOCOL, Feb. 12, 2009 (Published online) pp. 309-324). Itis also disclosed that cells have different functions depending on thesize of a spheroid (see, Franziska Hirschhaeuser, et al., “Multicellulartumor spheroids: An underestimated tool is catching up again”, Journalof Biotechnology 148, 2010, pp. 3-15, and C'ELINE LIU BAUWENS, et al.,“Control of Human Embryonic Stem Cell Colony and Aggregate SizeHeterogeneity Influences Differentiation Trajectories”, STEM CELL, 2008,pp. 2300-2310). In addition to the technique of forming a spheroid asmentioned above, a technique of controlling the size of a spheroid hasattracted attention. Further, since it is possible to reproduce aspecific function of a cell, it is expected that this technique ofcontrolling the size of a spheroid will be applicable in various fields,for example, the artificial organ and bioreactor fields. In suchapplications, a technique of preparing a large number of spheroids andrecovering the spheroids is important.

As means for creating a spheroid having a uniform diameter, JapaneseUnexamined Patent Application Publication No. H08-131153 discloses amethod of controlling the size of each spheroid formed by changing thenumber of cells to be seeded in a 96WP with a U-shaped bottom on which ahydrophilic membrane is formed. However, the number of spheroids perculture area is small, and thus it is difficult to prepare a largenumber of spheroids. As other methods for creating a spheroid having auniform diameter, Japanese Unexamined Patent Application Publication No.2010-88347, International Patent Publication No. WO 2012/036011, andInternational Patent Publication No. WO 2013/042360 disclose methods offorming a spheroid in a micro-space.

However, in the culture method disclosed in Japanese Unexamined PatentApplication Publication No. H08-131153, the culture efficiency isextremely low, which is a rate-limiting step for the large-scaleculture. In the culture methods disclosed in Japanese Unexamined PatentApplication Publication No. 2010-88347 and International PatentPublication No. WO 2012/036011, the efficiency of formation of spheroidsper unit area is high, but there is a possibility that the spheroidswill be removed from the inside of the culture space during replacementof the medium. Accordingly, careful attention is required duringreplacement of the medium. Moreover, a study has been made on a methodof causing a part of a spheroid to adhere to the inside of a micro-spaceso as to prevent removal of the spheroid (International PatentPublication No. WO 2013/042360). However, since adhesion property isdifferent in each type of cell, it is necessary to consider a surfacetreatment method for each of the cells to be used, and thus the methodis impractical.

SUMMARY

The present invention has been made in view of the above-mentionedbackground. An object of the present invention is to design amicro-space structure which facilitates replacement of a medium andharvesting of cells, and to provide a culture chamber having the saidmicro-space structure, and a culture method using the said culturechamber, to make it possible to prepare spheroids with a uniform sizewith high efficiency, or to prepare a large number of spheroids with auniform size with high efficiency.

According to an aspect of the present invention, a culture chamberaccording to one embodiment includes a plurality of recesses each formedof a bottom portion and an opening portion. The bottom portion has oneof a hemispherical shape and a truncated cone shape. The opening portionis defined by a wall that surrounds an area from a boundary between theopening portion and the bottom portion to an end of each of therecesses, the wall having a taper angle in a range from 1 degree to 20degrees. In addition, an equivalent diameter of the boundary is in arange from 50 μm to 2 mm and a depth from a bottom of the bottom portionto the end of each of the recesses is in a range from 0.6 or more timesto 3 or less times the equivalent diameter. The wall defining theopening portion forms a surface continuous to the bottom portion, and aninclination of the continuous surface changes at the boundary.

In the culture chamber according to one embodiment, it is preferablethat the end of each of the recesses have one of a hemispherical shape,a trapezoidal shape, and an inverted triangular shape. It is alsopreferable that an area between two adjacent recesses be flat and adistance between the two recesses be in a range from 5 μm to 50 μm.

Further, in the culture chamber according to one embodiment, it ispreferable that the culture chamber be a resin molding formed of one ora combination of two or more selected from the group consisting ofacrylic resin, polylactic acid, polyglycolic acid, styrene resin,acrylic styrene copolymer resin, polycarbonate resin, polyester resin,polyvinyl alcohol resin, ethylene vinyl alcohol copolymer resin,thermoplastic elastomer, vinyl chloride resin, and silicon resin. It ispreferable that a functional group be formed on the recesses by asurface modification treatment method of any one of plasma treatment,glass coating, corona discharge, and UV ozonation, or a combinationthereof and the treatment be performed so that a water contact anglebecomes 45 degrees or less.

It is preferable that a hydrophilic polymer chain that inhibits celladhesion be immobilized in the recesses.

It is preferable that a phospholipid or a phospholipid-polymer complexbe immobilized in the recesses.

It is preferable that the recesses each have a cell non-adhesive surfaceon which at least one polymer of a hydrophilic polymer chain thatinhibits cell adhesion, and a phospholipid, or a phospholipid-polymercomplex is immobilized after a functional group is formed in therecesses by a surface modification treatment method of any one of plasmatreatment, glass coating, corona discharge, and UV ozonation, or acombination thereof and the treatment is performed so that a watercontact angle becomes 45 degrees or less.

It is preferable that hydrophilic polymer chain be poly(hydroxyethylmethacrylate), and it is more preferable that an average molecularweight of the poly(hydroxyethyl methacrylate) be 100,000 or more.

According to an aspect of the present invention, a culture methodaccording to one embodiment uses any one of the culture chambersdescribed above. This culture method includes: dispersing cells into amedium, a total number of the cells being equal to or greater than anumber (N) of the recesses of the culture chamber and equal to or lessthan a number obtained by multiplying the number (N) of the recesses bya value obtained by dividing a volume (V1) of a space defined by each ofthe recesses by a volume (V2) of cells to be seeded; and adding themedium to the culture chamber.

In one aspect of the culture method according to an embodiment of thepresent invention, it is preferable that one spheroid be formed in onespace defined by each of the recesses, and it is more preferable that aspheroid be formed in the space and the spheroid is allowed to grow(proliferate).

In the case of differentiating and inducing a spheroid, the spheroid ispreferably induced in a state where the spheroid is formed in the space.

It is preferable that 60% or more of a total number of spheroids formedin the culture chamber have a diameter in a range of ±5% of an averagespheroid diameter.

It is preferable that cells in the recesses be recovered by agitatingthe medium, and it is more preferable that the agitation of the mediumbe done by any one of the following means: agitation of the medium byshaking the culture chamber; agitation of the medium by sucking anddischarging the medium; agitation of the medium by disposing a stirringblade in the culture chamber; and agitation of the medium by placing astirrer in the culture chamber, or a combination thereof.

It is preferable that the medium be replaced at least once and 20% ormore of the medium be replaced.

According to another aspect of the present invention, a culture methodaccording to one embodiment uses any one of the culture chambersdescribed above. The culture method for cell seeding, cell culture,replacement of a medium, and harvesting of cells includes the steps of:a) dispersing cells into a medium, the number of the cells being equalto or greater than a number (n) of recesses of the culture chamber andequal to or less than a number obtained by multiplying the number (n) ofthe recesses by a value obtained by dividing a volume (V) of each of therecesses by a volume (v) of cells to be seeded, and adding the medium tothe culture chamber; b) culturing the cells in the culture chamber for12 hours or more to form a spheroid; c) sucking 20% or more of themedium and then injecting the same amount of fresh medium; d) repeatingthe steps a) to c) a plurality of times to allow the spheroid to grow;e) allowing the spheroid to grow to a desired size and then agitatingthe medium to suspend the cells within the recesses in the medium; andf) sucking the medium including the cells by a suction machine torecover the cells.

According to the present invention, it is possible to provide a culturechamber capable of preparing a large number of spheroids with a uniformsize with high efficiency and having a micro-space structure which isdesigned to enable replacement of a medium and harvesting of cells, anda culture method using the culture chamber.

Additional features and advantages of the embodiments disclosed hereinwill be set forth in the detailed description which follows, and in partwill be readily apparent to those skilled in the art from thatdescription or recognized by practicing the disclosed embodiments asdescribed herein, including the detailed description which follows, theclaims, as well as the appended drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing an example of a culture chamber according toan embodiment;

FIG. 2 is a cross-sectional view showing an example of the shape of arecess according to a first embodiment;

FIG. 3 is a top view showing an example of the shape of the recessaccording to the first embodiment;

FIG. 4 is a diagram showing an example of the shape of a recess formedusing a part of a spherical shape according to a second embodiment;

FIG. 5 is a diagram showing another example of the shape of a recessformed using a part of a spherical shape according to the secondembodiment;

FIG. 6 is a diagram showing an example of the shape of a recess formedusing a truncated cone shape according to the second embodiment;

FIG. 7 is a diagram showing another example of the shape of a recessaccording to the second embodiment;

FIG. 8 is a diagram showing an example of the shape of an openingportion according to a third embodiment;

FIG. 9 is a diagram showing another example of the shape of an openingportion according to the third embodiment;

FIG. 10 is a diagram showing an example of the structure of a culturechamber according to a fourth embodiment;

FIG. 11 is a diagram showing another example of the structure of theculture chamber according to the fourth embodiment;

FIG. 12 is a diagram showing still another example of the structure ofthe culture chamber according to the fourth embodiment;

FIG. 13 is a diagram showing a survival rate of spheroids in an exampleand a survival rate of spheroids in a comparative example at the time ofreplacement of a medium;

FIG. 14 shows a photograph illustrating an image of spheroids in theexample and a photograph illustrating an image of spheroids in thecomparative example before and after the replacement of a medium;

FIG. 15 shows a photograph illustrating an image of cells before thecells are recovered in the example and a photograph illustrating animage of cells after the cells are recovered in the example; and

FIG. 16 shows photographs of spheroids recovered from a culture chamberaccording to the example.

DETAILED DESCRIPTION

Reference will now be made in detail to the present preferredembodiments of the present disclosure, examples of which are illustratedin the accompanying drawings. Whenever possible, the same referencenumerals will be used throughout the drawings to refer to the same orlike parts. However, this disclosure may be embodied in many differentforms and should not be construed as limited to the embodiments setforth herein.

First Embodiment

<Culture Chamber>

FIG. 1 is a diagram showing an example of a culture chamber according toan embodiment. FIG. 1 shows a part of a culture plate 3 including aplurality of culture chambers 1. FIG. 1 shows a part of a culture plate3 including a plurality of culture chambers 1. The upper part of FIG. 1shows some of a plurality of recesses 10 which are formed in the bottomof each of the culture chambers 1, when viewed from the top of theculture plate 3. The plurality of recesses 10 are arranged in each ofthe culture chambers 1. In terms of the production of the culturechambers 1 and the efficiency of cell culture, it is preferable toarrange the plurality of recesses 10 in a regular manner. One culturechamber 1 corresponds to, for example, one well arranged in a plateincluding a plurality of wells. In other words, the plurality ofrecesses 10 are arranged in the respective wells of a well plate.

A well plate is an experimental/testing instrument formed of a flatplate having a number of dents (holes or wells), and each well is usedas a test tube or a petri dish. The number of wells is, for example, 6,24, 96, 384, or more. Examples of the shape of the bottom of each wellinclude a flat shape, a round shape, and a combination of a number ofelongated microtubes (deep well plate).

Each recess 10 forms a micro-space, which is a small space for cultureof cells, and thus each recess can also be referred to as amicrochamber.

FIGS. 2 and 3 show an example of the shape of a recess according to afirst embodiment. FIG. 2 shows a cross-sectional view of one recess 10,and FIG. 3 shows a top view of one recess 10. The recess 10 shown inFIG. 3 is an example of the detailed structure of each of the recesses10 shown in the upper part of FIG. 1 .

Each recess 10 is composed of a bottom portion 11 and an opening portion12. The bottom portion 11 is a portion serving as the bottom of theculture chamber 1, and the opening portion 12 is a portion disposedabove the bottom portion 11. A portion where the bottom portion 11 andthe opening portion 12 are in contact is referred to as a boundary. InFIG. 2 , a portion whose length is indicated by an arrow “R” correspondsto the location of the boundary. In FIG. 3 , the boundary location isindicated by a double dashed chain line. Note that the bottom portion 11and the opening portion 12 are formed of a continuous surface and areproduced in an integrated manner.

FIGS. 2 and 3 show an equivalent diameter R and a depth (height) H ofeach of the plurality of recesses 10 formed in the culture chamber 1.

The term “equivalent diameter R” refers to the diameter of a circleinscribed in the bottom portion 11 of each recess 10. In this case, theequivalent diameter R is the diameter of an inscribed circle that isinscribed at the boundary between the bottom portion 11 and the openingportion 12. More specifically, the equivalent diameter R is the diameterof a circle inscribed in a shape of a plane that is perpendicular to thedirection of the height H of each recess 10 at the boundary.

The term “depth D” refers to a length from the bottom on the inside ofthe bottom portion 11 to an upper end of each recess 10. The upper endof the recess 10 corresponds to an end (upper end) of the openingportion 12. The depth D corresponds to the depth of a space formed bythe recess 10. In other words, the depth D is a depth from the bottom ofa space, which is formed by the bottom portion 11, to an upper end of aspace formed by the opening portion 12. FIG. 2 shows not only the depthD of the recess 10, but also a depth D1 of the bottom portion 11 and adepth D2 of the opening portion 12.

The bottom portion 11 forms a space (first space) in which cells arecultured. The bottom portion 11 has, for example, a hemispherical shape.For example, a shape obtained by dividing a spherical shape having theequivalent diameter R as a diameter into halves can be used. The shapeof the bottom portion 11 is not limited to a hemispherical shape. Otherspecific examples of the shape will be described in a second embodiment.

The opening portion 12 forms a space (second space) that operates tosupport culture and harvesting of cells. The opening portion 12 isformed of a wall which surrounds an area from a boundary between theopening portion 12 and the bottom portion 11 to an end (tip) of therecess 10 and which has a taper angle in a range from 1 degree to 20degrees. The taper angle of the wall constituting the opening portion 12is preferably in a range from 5 degrees to 15 degrees, and morepreferably, 10 degrees. This is because if the taper angle is extremelysmall, it is difficult to transfer cells from the recesses into a mediumduring harvesting of the cells, and if the taper angle is extremelylarge, the cells are removed during replacement of the medium.

Taper angles are represented by θ1 and θ2 in FIG. 2 . In an example ofthe shape of each recess 10 shown in FIGS. 2 and 3 , the taper angles θ1and θ2 are substantially the same.

The boundary between the bottom portion 11 and the opening portion 12 isformed in such a manner that the equivalent diameter R is in a rangefrom 50 μm to 1 mm. To supply nutrients to a central portion of aspheroid, the equivalent diameter is preferably in a range from 50 μm to500 μm, and more preferably, in a range from 100 μm to 500 μm. This isbecause it is said that nutrients and oxygen are transferred into cellsonly by diffusion and a central portion of a spheroid with a size of 300μm or less does not become necrotic (Efrem Curcio et al., “Mass transferand metabolic reactions in hepatocyte spheroids cultured in rotatingwall gas-permeable membrane system”, Biomaterials 28 (2007) 5487-5497).Accordingly, the above-mentioned diameter range is preferable to preventa spheroid from growing to the size of 300 μm.

On the contrary, when it is intended to cause necrosis in a centralportion of a cell, like in a cancer cell (Franziska Hirschhaeuser etal., “Multicellular tumor spheroids: An underestimated tool is catchingup again”, Journal of Biotechnology 148 (2010) 3-15, FIG. 1), theequivalent diameter R is preferably equal to or more than 400 μm andless than 2 mm. This is because, as mentioned above, nutrients can betransferred to a central portion of a spheroid with a size of 300 μm, sothat necrosis does not occur. Accordingly, in order to obtain a spheroidhaving a diameter of 300 μm or more, it is necessary that the equivalentdiameter be equal to or more than 400 μm.

In addition, the depth D from the bottom of the bottom portion to theend of each of the recesses is set in a range from 0.6 or more times to3 or less times the equivalent diameter R. The depth D is preferably ina range from 0.7 or more times to 1.2 or less times the equivalentdiameter R, and more preferably, in a range from 0.8 to 1 times theequivalent diameter R.

In each culture chamber 1, the area between two adjacent recesses 10 ispreferably flat. For example, the distance between two recesses 10 ispreferably in a range from 5 μm to 50 μm. This is because it ispreferable to increase the number of spheroids per unit area and culturethe spheroids at a high density so that a large number of spheroids canbe efficiently obtained. To achieve this, the area of the upper surfaceof the wall on which no spheroid is formed is preferably small as muchas possible. In this case, however, when the taper angle is small andthe wall is thin, cracking may easily occur due to a vibration duringcell seeding or replacement of a medium. Accordingly, the distancebetween two recesses is preferably 5 μm or more. In view of this, thedistance between two recesses is preferably in a range from 5 to 20 μm.

On the other hand, the two adjacent recesses 10 may come into contactwith each other. For example, a part of an end of one of the tworecesses 10 and a part of an end of the other one of the two recesses 10may come into contact with each other, so that the inclined surfaces ofthe opening portions 12, each of which forms a taper angle, may comeinto contact with each other to form a chevron shape.

The culture chamber 1 having the above-described shape is preferablyproduced in the following manner.

Each culture chamber 1 is preferably a resin molding formed of one or acombination of two or more selected from the group consisting of acrylicresin, polylactic acid, polyglycolic acid, styrene resin, acrylicstyrene copolymer resin, polycarbonate resin, polyester resin, polyvinylalcohol resin, ethylene vinyl alcohol copolymer resin, thermoplasticelastomer, vinyl chloride resin, and silicon resin.

A functional group is preferably formed on the recesses 10 of theculture chamber 1 by a surface modification treatment method of any oneof plasma treatment, glass coating, corona discharge, and UV ozonation,or a combination thereof and the treatment is preferably performed sothat the water contact angle becomes 45 degrees or less.

In addition, a hydrophilic polymer chain that inhibits cell adhesion ispreferably immobilized in the recesses 10. More preferably, thehydrophilic polymer chain is immobilized in the recesses 10 that aretreated so that the above-mentioned water contact angle becomes 45degrees or less.

Furthermore, a phospholipid or a phospholipid-polymer complex ispreferably immobilized in the recesses 10. More preferably, thisimmobilization treatment is performed on each recess 10 that is treatedso that the above-mentioned water contact angle becomes 45 degrees orless, each recess 10 in which a hydrophilic polymer chain isimmobilized, or a combination of these recesses 10.

Moreover, each of the recesses 10 preferably has a cell non-adhesivesurface on which at least one polymer of a hydrophilic polymer chainthat inhibits cell adhesion, and a phospholipid, or aphospholipid-polymer complex is immobilized after a functional group isformed in the recesses by a surface modification treatment method of anyone of plasma treatment, glass coating, corona discharge, and UVozonation, or a combination thereof and the treatment is performed sothat the water contact angle becomes 45 degrees or less. Morepreferably, this treatment is carried out together with one or acombination of the above-mentioned treatments.

The above-mentioned hydrophilic polymer chain is preferablypoly(hydroxyethyl methacrylate). More preferably, the average molecularweight of poly(hydroxyethyl methacrylate) is 100,000 or more.

<Culture Method>

Next, a method of culturing cells using the culture chambers 1 shown inFIGS. 1 to 3 will be described.

The cell culture is performed by the following steps: a) adding a mediumin which cells are dispersed to the culture chambers 1; b) culturing thecells; c) replacing the medium; d) allowing spheroids to grow; e)suspending the spheroids in the medium; and f) recovering the cells.

The above-mentioned steps can be classified into two steps, i.e., thestep of culturing cells (cell culture step) and the step of recoveringcells (cell harvesting step). The cell culture step includes the stepsa) to d), and the cell harvesting step includes the steps e) and f).

The term “spheroid” used herein refers to a three-dimensional cellcluster including a number of aggregated cells.

Each of the steps will be described below.

a) Step of Adding a Medium in which Cells are Dispersed to the CultureChambers 1

This step is a step of preparing for culture of cells. In this step, thetotal number of cells as described below are dispersed in a medium andare added to each culture chamber 1.

A lower limit of the total number of cells is equal to or greater thanthe number (n) of recesses 10 present in the culture chamber 1.

An upper limit of the total number of cells is equal to or less than anumber obtained by multiplying the number (n) of recesses by a valueobtained by dividing the volume (V) of each of the recesses 10 of theculture chamber 1 by the volume (v) of cells to be seeded. The upperlimit of the total number of cells can be represented by the followingformula using symbols: V/v×n. This is based on the premise that thevolumes (V) of the plurality of recesses 10 are the same. If the volumes(V) of the plurality of recesses 10 are different, an average value isused.

The medium is adjusted depending on the cells to be cultured.

b) Step of Culturing Cells

The cells are cultured for 12 hours or more in each culture chamber 1 tothereby allow the cells to form a spheroid. When the medium is added toeach culture chamber 1, the cells dispersed in the medium are loadedinto the recesses 10 and the cells are cultured in the respectiverecesses 10. It is preferable to load one cell into each recess 10, andit is preferable to form one spheroid in the space formed by the bottomportion 11. In each recess 10, a cell proliferates at the bottom portion11 of the recess 10. If at least one cell is not present in each recessduring culture and seeding, no spheroid is formed in the recess 10,because any cell does not move from the adjacent recess 10 to the saidrecess 10 during culture. In order to culture spheroids at a highdensity, it is preferable to form a spheroid in each recess 10.Accordingly, it is preferable to form at least one cell in each recess10. In terms of the production efficiency, it is preferable to reducethe initial number of cells as much as possible and to recover as manyspheroids as possible, and therefore it is preferable that the number ofcells present in each recess 10 be small as much as possible. For thisreason, it is preferable that one cell be present in each recess 10.

c) Step of Replacing the Medium

During replacement of the medium, 20% or more of the medium in eachculture chamber 1 is sucked, and then the same amount of fresh medium isinjected into the culture chamber. It is preferable to replace themedium at least once during cell culture.

d) Step of Allowing Spheroids to Grow

The above-described steps a) to c) are performed a plurality of times tothereby allow spheroids to grow. In the case of differentiating andinducing spheroids, it is preferable that each spheroid be allowed togrow to a size limited by the space formed by the bottom portion 11 ofeach recess 10 and then the medium be replaced with differentiationinduction medium to thereby differentiate each spheroid. In addition, itis more preferable that 60% or more of the total number of spheroidsformed in the culture chambers 1 have a diameter in a range of ±5% of anaverage spheroid diameter.

e) Step of Suspending the Spheroids in the Medium

After each spheroid is grown to a desired size, the cells cultured ineach recess 11 are suspended in the medium by agitating the medium ineach culture chamber 1. For example, this step is carried out byagitating the medium. Specifically, the agitation of the medium can bedone by any one of the following means: (1) agitation of the medium byshaking each culture chamber 1; (2) agitation of the medium by suckingand discharging the medium (pipetting); (3) agitation of the medium bydisposing a stirring blade in each culture chamber 1; (4) agitation ofthe medium by placing a stirrer in each culture chamber 1; and (5)agitation of the medium by a combination of two or more of theabove-mentioned means (1) to (4).

f) Step of Recovering the Cells

The medium including the cells in each culture chamber 1 is sucked by asuction machine, to thereby recover the cells (spheroids) suspended inthe medium.

As described above in the first embodiment, seeding of cells,replacement of a medium, and harvesting of cells can be performed in thesame chamber, and in addition, spheroids can be recovered from eachculture chamber.

Culture of cells using the culture chambers 1 of the first embodimentenables formation of a spheroid having a desired size on the bottomportion 11. Further, the cultured spheroids can be efficientlyrecovered. Specifically, the structure of each recess 10 including thebottom portion 11 and the opening portion 12 makes it possible to easilymaintain a state in which cells adhering to the bottom portion 11 orbeing suspended in the medium are prevented from being removed when themedium is sucked during replacement of the medium. Thus, it can beexpected that the removal of cells from the bottom portion 11 issuppressed. On the other hand, during the harvesting of cells, when themedium in the bottom portion 11 is sucked and discharged, it can beexpected that the medium is allowed to easily flow through the openingportion 12. It can also be expected that the use of the hemisphericalshape of the bottom portion 11 contributes to the formation of spheroidswith a uniform shape and size.

Second Embodiment

While an example of the structure in which the bottom portion 11 has ahemispherical shape has been described in the first embodiment, othershapes of the bottom portion will be described in a second embodiment.The bottom portion may have any form, such as a shape formed using apart of a spherical shape, a truncated cone shape, or a linear shape.The linear shape of the bottom portion is a form having no substantialbottom portion and having a recess formed only by an opening portion.FIGS. 4 to 7 show examples of the shape of each recess according to thisembodiment. FIGS. 4 to 7 show recesses 20A to 20D having bottom portions21A to 21D, respectively, which are different from the bottom portion 11of the first embodiment. Since the opening portion 12 can be formed withthe same shape as that of the first embodiment, FIGS. 4 to 7 showexamples of the shape of each recess in which the opening portionshaving the same shape are combined.

While in the first embodiment, a hemispherical shape, which is a shapeobtained by dividing a spherical shape into halves, is used for thebottom portion 11, FIGS. 4 and 5 show examples in which differenthemispherical shapes are used for the bottom portion. FIG. 4 shows thebottom portion 21A for which a portion less than a half of a sphericalshape is used. In other words, FIG. 4 shows a case where a part of ahemispherical shape is used for the bottom portion 21A. FIG. 5 shows thebottom portion 21B having a cylindrical shape with a hemisphericalbottom. In the case of the shape of the bottom portion 21B shown in FIG.5 , as the length of the cylindrical portion increases, the cells areless likely to be suspended into the medium from the bottom portion 21Bduring harvesting of the cells. Accordingly, it is preferable to adjustthe length of the cylindrical portion. For example, it is preferable toform the bottom portion 21B and the opening portion 12 so as to maintainthe same ratio (1:1) between the depth (height) of the bottom portion21B and the depth (height) of the opening portion 12.

FIG. 6 shows the bottom portion 21C for which a truncated cone shape isused. When the bottom portion is flat, the reflection and interferenceof light can be reduced, and thus it is useful for observation with amicroscope.

FIG. 7 shows an example of the shape of the recess 20D in which thebottom portion 21D has a linear shape, i.e., the bottom portion 21D doesnot form a space. The efficiency of culture and harvesting of cells inthe recess 20D is lower than that of culture chambers having othershapes. However, the recess 20D has an advantage in facilitating theproduction process for the culture chambers.

A case where the opening portion 12 is formed with a shape similar tothat of the first embodiment has been described in this embodiment.However, the present invention is not limited to this case.

The method of culturing cells using the culture chambers according tothis embodiment is similar to that of the first embodiment, and thus thedescription thereof is omitted.

The culture chambers according to this embodiment can provide the sameadvantageous effects as those of the first embodiment.

Third Embodiment

A mode in which the shape of the opening portion 12 is a circular shapeor a substantially circular shape has been described in the aboveembodiments. A culture chamber including opening portions each having ashape other than a circular shape or a substantially circular shape willbe described. An end of each opening portion may have a shape other thana circular shape or a substantially circular shape, such as ahemispherical shape, a trapezoidal shape, or an inverted triangularshape. On the other hand, it is necessary that the shape of the boundarywhere the opening portion contacts the bottom portion (the boundaryportion of the opening portion) be the same as the shape of the boundaryportion of the bottom portion. FIGS. 8 and 9 show recesses 30A and 30B,respectively, each having an end with a shape different from that of theopening portion 12 of the first embodiment. While FIGS. 8 and 9 show thesame bottom portion 11 as that of the first embodiment, a combination ofany of the bottom portions 21A to 21D of the second embodiment, or abottom portion having another shape may be used. The bottom portion andthe opening portion may have any shape as long as an inclined surfacecan be formed continuously at the boundary between the bottom portionand the opening portion.

FIG. 8 shows an example of the shape of an end of the opening portion32A that is formed in a curve. FIG. 8 is a top view of the recess 30A.An end of the bottom portion 11 is indicated by a circle having theequivalent diameter R, and the outer periphery of the opening portion32A is indicated by a curve. The end of the opening portion 32A has acurved shape which is not symmetric in the horizontal direction and thevertical direction. However, the end of the opening portion 32A may havea shape which is symmetric in the horizontal direction or the verticaldirection. FIG. 9 shows an example in which an end of the openingportion 32B has a rectangular shape. Although FIG. 9 shows an example inwhich an end of the opening portion has a square shape, the end of theopening portion may have another polygonal shape, or a combination of acurve and a straight line. FIG. 9 is a top view of the recess 30B. Theend of the bottom portion 11 is indicated by a circle having theequivalent diameter R, and the outer periphery of the opening portion32B is indicated by a solid square. For example, the shape of the end ofthe bottom portion may be modified so as to adjust the area of the spacebetween the end of the bottom portion and the adjacent recess. Since itis necessary for the shape of the end of the opening portion to play arole of promoting the suspension of cells, the taper angle is important.

In the shape examples shown in FIGS. 8 and 9 , the taper angle has avalue that varies depending on the shape of the opening portions 32A and32B. This is because the inclination of the inclined surface that formsthe wall varies depending on the shape of the opening portions 32A and32B.

Each of the shapes of the opening portions illustrated in thisembodiment can be combined with the shape of the bottom portion 11described in the first embodiment, or the shape of the bottom portiondescribed in the second embodiment. In addition, these shapes can alsobe combined with a shape other than the shapes of the bottom portionillustrated in the above embodiments, as a matter of course.

The method of culturing cells using the culture chambers according tothis embodiment is similar to that of the first embodiment, and thus thedescription thereof is omitted.

The culture chambers according to this embodiment can provide the sameadvantageous effects as those of the first embodiment.

Fourth Embodiment

FIG. 1 illustrates a mode in which the culture chambers 1 according toone embodiment are arranged in the culture plate 3 (well plate). Theculture chambers 1 according to one embodiment can also be formed in achamber (instrument) other than the culture plate 3 shown in FIG. 1 .FIGS. 10 to 12 show examples of the structure of a culture chamberaccording to a fourth embodiment. FIG. 10 is a schematic view showing anexample of the structure in which a flask-shaped culture flask is used.FIG. 11 is a schematic view showing an example of the structure in whicha frame of a culture plate is used. FIG. 12 is a schematic view showingan example of the structure in which the culture plate shown in FIG. 11is designed in a stack shape and used.

In FIG. 10 , a bottom surface of a culture flask 4 is used as a culturesurface 4A (culture bottom surface). The culture surface 4A correspondsto each culture chamber 1 shown in FIG. 1 . Accordingly, the culturesurface 4A can also be referred to as a culture chamber. Like eachculture chamber 1 shown in FIG. 1 , the culture surface 4A is a unit forusing the same medium. The culture flask 4 includes a cap 4B. The areaof the culture surface 4A can be designed depending on the intended use.Examples of the size of a typical culture flask include 25, 75, and 225cm². A plurality of recesses 40 are formed in the culture surface 4A ofthe culture flask 4. For example, in the bottom surface of the cultureflask 4, a shaded area is designed as the culture surface 4A and theplurality of recesses 40 are formed in the culture surface 4A. The shapeof each recess 40 (the shape of each of the bottom portion and theopening portion) may be any one of the shapes illustrated in the aboveembodiments.

FIG. 11 shows an example in which only the frame of a culture plate isused. In FIG. 1 , the culture chambers 1 (wells) are formed in theculture plate 3, whereas in FIG. 11 , a bottom surface of a cultureplate 5 is used as a culture surface 5A (culture bottom surface). Theculture surface 5A corresponds to each culture chamber 1 shown in FIG. 1. Accordingly, the culture surface 5A can also be referred to as aculture chamber. The culture surface 5A is a unit for using the samemedium. The lower part of FIG. 11 shows an example (schematiccross-sectional view) of the structure of the culture surface 5A. Forexample, in the bottom surface of the culture plate 5, a shaded area isdesigned as the culture surface 5A and a plurality of recesses 50 areformed in the culture surface 5A. The recesses 50 shown in FIG. 11 areschematically illustrated. The number, size, and the like of therecesses 50 are designed depending on the intended use. The shape ofeach recess 50 (the shape of each of the bottom portion and the openingportion) may be any one of the shapes illustrated in the aboveembodiments.

FIG. 12 shows a structural example of a cell stack form in which aplurality of culture plates 5 shown in FIG. 11 are stacked. In otherwords, FIG. 12 shows an example of a multi-stage structure. In a casewhere cells are cultured in a closed system with a larger area, the cellstack form is generally used. While FIG. 12 illustrates an example inwhich the culture plates 5 shown in FIG. 11 are stacked, the cultureplates 3 shown in FIG. 1 may be stacked. In FIG. 12 , the illustrationof a chamber that accommodates the plurality of stacked culture chambersand provides a mechanism for replacement of a medium is omitted. Forexample, a culture chamber having a typical stack shape can be used asthe chamber that accommodates the plurality of culture plates. Theexplanation thereof is herein omitted.

Other Embodiments

In the above embodiments, the boundary between the bottom portion andthe opening portion is defined to be parallel to the bottom of eachculture chamber. However, it is not necessary that the boundary beparallel to the bottom. For example, the boundary may be inclined withrespect to the bottom, or may be formed in a curve. It is only necessarythat a sufficient space to form a spheroid can be formed in the bottomportion 11.

Example

As for a culture chamber for culturing a cell aggregate and a harvestingmethod thereof, experiments were conducted according to the followingexample and comparative example.

(1) Culture Chamber

Culture chambers shown in Table 1 were used.

TABLE 1 Example Comparative Example Chamber A culture place inEZ-Sphere ® which patterns of (manufactured by shapes shown in ASAHIGLASS CO., FIGS. 1 to 3 are LTD.) arranged in a culture bottom surfacewas prepared. Bottom shape of hemispherical shape spherical shapemicro-chamber The number of 600 578 micro-spaces/wells Taper angle 10degrees There is no portion at which a taper angle is formed. Equivalent500 μm 400-500 μm diameter R Depth 400 μm (0.8 R) 150-200 μm Surfacep-HEMA a product with a surface on which a cell non-adhesion treatmentis performed Shape of chamber 24-well plate 24-well plate

As the culture chamber of the example, a culture plate in which wells(culture chambers 1) each including the recesses 10 shown in FIGS. 1 to3 are formed was prepared.

In Table 1, microchambers respectively correspond to the recesses 10shown in FIGS. 1 to 3 and each of micro-spaces is a space formed by eachrecess 10 (micro-space). It can be said that the number of micro-spacesper well is the number of recesses per well.

(2) Culture Method

To calculate a survival rate and a harvesting rate, which are describedlater, by image analysis, endodermal cells labeled with fluorescence ofGFP were used. The endodermal cells, vascular endothelial cells, andhuman mesenchymal stem cells were mixed at a ratio of 10:5-10:2, and thecells were cultured for 30 days in an endothelial cell medium kit-2:EGM-2 BulletKit (product code CC-3162: Lonza). The medium was replacedonce every two days.

(3) Measurement of the Survival Rate of Spheroids

All the wells were observed with a confocal laser microscope, andspheroids were recognized by image analysis software. Then, the numberof the recognized spheroids was counted and the number was determined asthe number of spheroids. The survival rate of spheroids was calculatedby the following expression.Spheroid survival rate (%)=(the number of spheroids)×100/(the number ofmicro-spaces)

A few hours after the cells were seeded (0th day), a spheroid-likecluster was formed in 90% or more of the micro-spaces in all the culturechambers. A value obtained by dividing the number of spheroids obtainedafter replacement of the medium on the 10th and 20th days of the cultureby the number of spheroids obtained on the 0th day was determined as thesurvival rate of spheroids.

(4) Harvesting Method

After completion of the culture, the solution was agitated with apipette (manufacturer, model number), and the suspended spheroids wererecovered. For example, a pipette capable of sucking 1 mL of medium atmaximum is suitably used for a 24-well plate that contains 500 μL to 1mL of medium.

(5) Harvesting Efficiency

Before and after the harvesting of spheroids, images of the spheroidswere taken by a confocal laser microscope.

(6) Results

FIG. 13 shows the survival rate of spheroids during replacement of themedium. The vertical axis represents the survival rate of spheroids(Sphere Number) and the horizontal axis represents the number of days ofculture.

FIG. 13 shows data obtained from the start of culture to the 20th day ofculture. As shown in FIG. 13 , the survival rate of spheroids in thecomparative example significantly decreased in comparison to that in theexample. After culture for 20 days, the survival rate of spheroids inthe example was 60% or more. This indicates that the survival rate ofspheroid in the example was improved 1.5 times as high as that in thecomparative example.

FIG. 14 shows the images of spheroids obtained before and afterreplacement of the medium in the example and the comparative example.FIG. 14 shows the images of spheroids in the culture chamber before andafter the second replacement of the medium on the fourth day of culture.The left side of FIG. 14 shows a photograph of the example (Kurarayp-HEMA), and the right side of FIG. 14 shows a photograph of thecomparative example (Iwaki MPC). The upper part of the figure showsimages taken before replacement of the medium, and the lower part (belowan arrow) of FIG. 14 shows images taken after replacement of the medium.More specifically, the images in the lower part of FIG. 14 show thestate after replacement of the medium was performed twice, i.e., half ofthe medium was replaced (replacement of half of the medium), from thestate before replacement of the medium.

In the images, white dots correspond to spheroids. Before replacement ofthe medium, spheroids are confirmed over the entire area. Afterreplacement of the medium, in the example, there is no large differencein the number of spheroids before and after replacement of the mediumand almost all the spheroids survived, whereas in the comparativeexample, only about a half of the spheroids were survived.

FIG. 15 shows images taken before and after the harvesting of cells inthe example. The left side (BEFORE) in FIG. 15 shows the image of cellstaken before the harvesting of cells, and the right side (AFTER) in FIG.15 shows the image of cells taken after the harvesting of cells. Theupper part of FIG. 15 shows the image of the entire culture chamber, andthe lower part of FIG. 15 shows the enlarged image of a part of theculture chamber. FIG. 16 shows photographs of the spheroids recoveredfrom the culture chamber according to the example.

Dot-like portions in each black circular microchamber (recess)correspond to spheroids. No dot-like portions are found in the imagetaken after the harvesting of cells, which indicates that almost 100% ofthe cells can be recovered. Further, as shown in FIG. 16 , the recoveredcells have an excellent spheroid shape, and each spheroid was notdestroyed by the harvesting operation.

Note that the present invention is not limited to the embodimentsdescribed above. Those skilled in the art can easily make modifications,additions, and conversions on each component in the above embodimentswithin the scope of the present invention.

REFERENCE NUMBER LISTING

-   -   1 CULTURE CHAMBER    -   3, 5 CULTURE PLATE    -   4A, 5A CULTURE SURFACE    -   5 CULTURE FLASK    -   10, 20A-20D, 30A, 30B, 40, 50 RECESS    -   11, 21A-21D BOTTOM PORTION    -   12, 32A, 32B OPENING PORTION

It will be apparent to those skilled in the art that variousmodifications and variations can be made to embodiment of the presentdisclosure without departing from the spirit and scope of thedisclosure. Thus it is intended that the present disclosure cover suchmodifications and variations provided they come within the scope of theappended claims and their equivalents.

What is claimed is:
 1. A culture chamber comprising: a plurality ofrecesses, each recess formed of a bottom portion and an opening portion,wherein: the bottom portion having a depth D1 comprises a hemisphericalshape or a truncated cone shape, the opening portion having a depth D2is defined by a wall that surrounds an area from a boundary between theopening portion and the bottom portion to an end of each of therecesses, the wall comprising a taper angle of 1 degree to 20 degrees,an equivalent diameter of the boundary is from 50 μm to 1 mm and a depthD from a bottom of the bottom portion to the end of each of the recessesis from 0.6 or more times to 3 or less times of the equivalent diameter,wherein the depth D equals D1+D2, the wall defining the opening portionforms a surface continuous to the bottom portion, and an inclination ofthe surface continuous to the bottom portion changes at the boundary,and wherein an area between two adjacent recesses is flat and a distancebetween the two recesses is in a range from 5 μm to 50 μm.
 2. Theculture chamber according to claim 1, wherein the end of each of therecesses comprises one selected from the group consisting of ahemispherical shape, a trapezoidal shape, and an inverted triangularshape.
 3. The culture chamber according to claim 1, wherein the culturechamber is a resin molding formed of one or a combination of two or moreselected from the group consisting of an acrylic resin, a polylacticacid, a polyglycolic acid, a styrene resin, an acrylic styrene copolymerresin, a polycarbonate resin, a polyester resin, a polyvinyl alcoholresin, an ethylene vinyl alcohol copolymer resin, a thermoplasticelastomer, a vinyl chloride resin, and a silicon resin.
 4. The culturechamber according to claim 1, wherein a functional group is formed onthe recesses by a surface modification treatment method selected fromthe group consisting of plasma treatment, glass coating, coronadischarge, UV ozonation, and a combination thereof and the treatment isperformed so that a water contact angle becomes 45 degrees or less. 5.The culture chamber according to claim 1, wherein a hydrophilic polymerchain that inhibits cell adhesion is immobilized in the recesses.
 6. Theculture chamber according to claim 1, wherein a phospholipid or aphospholipid-polymer complex is immobilized in the recesses.
 7. Theculture chamber according to claim 1, wherein each of the recessescomprises a cell non-adhesive surface on which at least one polymer of ahydrophilic polymer chain that inhibits cell adhesion, and aphospholipid, or a phospholipid-polymer complex is immobilized after afunctional group is formed in the recesses by a surface modificationtreatment method selected from the group consisting of plasma treatment,glass coating, corona discharge, UV ozonation, and a combinationthereof, and the treatment is performed so that a water contact anglebecomes 45 degrees or less.
 8. The culture chamber according to claim 7,wherein the hydrophilic polymer chain is poly(hydroxyethylmethacrylate).
 9. The culture chamber according to claim 8, wherein anaverage molecular weight of the poly(hydroxyethyl methacrylate) is100,000 or more.
 10. The culture chamber according to claim 2, whereinthe culture chamber is a resin molding formed of one or a combination oftwo or more selected from the group consisting of acrylic resin,polylactic acid, polyglycolic acid, styrene resin, acrylic styrenecopolymer resin, polycarbonate resin, polyester resin, polyvinyl alcoholresin, ethylene vinyl alcohol copolymer resin, thermoplastic elastomer,vinyl chloride resin, and silicon resin.
 11. The culture chamberaccording to claim 2, wherein a functional group is formed on therecesses by a surface modification treatment method of any one of plasmatreatment, glass coating, corona discharge, and UV ozonation, or acombination thereof and the treatment is performed so that a watercontact angle becomes 45 degrees or less.
 12. The culture chamberaccording to claim 2, wherein a hydrophilic polymer chain that inhibitscell adhesion is immobilized in the recesses.
 13. The culture chamberaccording to claim 2, wherein a phospholipid or a phospholipid-polymercomplex is immobilized in the recesses.
 14. The culture chamberaccording to claim 13, wherein each of the recesses has a cellnon-adhesive surface on which at least one polymer of a hydrophilicpolymer chain that inhibits cell adhesion, and a phospholipid, or aphospholipid-polymer complex is immobilized after a functional group isformed in the recesses by a surface modification treatment method of anyone of plasma treatment, glass coating, corona discharge, and UVozonation, or a combination thereof and the treatment is performed sothat a water contact angle becomes 45 degrees or less.
 15. The culturechamber according to claim 14, wherein the hydrophilic polymer chain ispoly(hydroxyethyl methacrylate).
 16. The culture chamber according toclaim 15, wherein an average molecular weight of the poly(hydroxyethylmethacrylate) is 100,000 or more.